Rat embryonic stem cell

• US 9,700,023 B2
• Filed: 12/18/2013
• Issued: 07/11/2017
• Est. Priority Date: 03/04/2004
• Status: Active Grant

• Current Assignee: Sumitomo Chemical Company Limited
• Original Assignee: DS Pharma Biomedical Co., Ltd. (DS Pharma Promo Co., Ltd.), National Cancer Center
• Inventors: Teratani, Takumi, Ochiya, Takahiro
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US14/132,969
• Publication Number:
  US 20140109247A1
• Time in Patent Office: 1,301 Days
• Field of Search: 800 21, 800 25, 435353, 435347, 435463
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention provides a rat embryonic stem cell characterized by having the following properties of (a) expressing Oct3/4 gene and Nanog gene, (b) positive for alkaline phosphatase activity, (c) having an embryoid body forming ability, (d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4, (e) having the same number of chromosomes as does a normal rat cell, (f) capable of being subcultured and holding the undifferentiated state, (g) having in vitro pluripotency, (h) having a potential to differentiate for cells of three embryonic germ lineages, (i) having teratoma formation ability, and (j) having an ability to produce a chimeric rat, a method of establishing the aforementioned rat embryonic stem cell and the like.

Claims

1. A method of producing a chimeric rat, which comprises performing a process comprising the following steps (X)-(Z):

(X) a step for introducing a desired gene into a rat embryonic stem cell,

(Y) a step for preparing a chimeric rat embryo for transplantation by (i) injecting the rat embryonic stem cell into which the gene was introduced into a blastocoele of a rat blastocyst or a morula stage- or 16-cell stage-rat embryo or (ii) co-culturing the rat embryonic stem cell into which the gene was introduced with two 8-cell stage-rat embryos without zona pellucidas,

(Z) a step for transferring the chimeric rat embryo for transplantation into the uterus of a pseudopregnant female rat to produce the chimeric rat, wherein the rat embryonic stem cell has the following properties (a)-(k);

(a) expressing Oct3/4 gene and Nanog gene,

(b) positive for alkaline phosphatase activity,

(c) having an embryoid body forming ability,

(d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4,

(e) having the same number of chromosomes as does a normal rat cell,

(f) capable of being subcultured and holding the undifferentiated state,

(g) having in vitro pluripotency,

(h) having a potential to differentiate for cells of three embryonic germ lineages,

(i) having teratoma formation ability,

(j) having an ability to produce a chimeric rat,

(k) being germline-competent,

wherein the rat embryonic stem cell is obtained by the method consisting essentially of the following steps (A)-(E) performed using a culture medium with 2% or less serum concentration;

(A) culturing a rat blastocyst on inactivated mouse embryonic fibroblasts in a leukemia inhibitory factor (LIF)-free culture medium to form an inner cell mass in the blastocyst,

(B) dissociating the inner cell mass, wherein the dissociated inner cell mass is in a cell aggregate state,

(C) culturing primary embryonic stem cells resulting from a culture of the dissociated inner cell mass on inactivated mouse embryonic fibroblasts until the primary embryonic stem cells can be passaged,

(D) dissociating the primary embryonic stem cells, which can be passaged, wherein the dissociated primary embryonic stem cells are in a cell aggregate state,

(E) culturing the dissociated primary embryonic stem cells on inactivated mouse embryonic fibroblasts to establish an embryonic stem cell,

wherein a rat leukemia inhibitory factor (rLIF)-containing culture medium is used in steps (C)-(E).

2. A method of producing a genetically modified rat, which comprises a step for crossing a chimeric rat with a wild-type rat to produce a heterozygous genetically modified rat, wherein the chimeric rat is produced by a process comprising the following steps (X)-(Z):

(X) a step for introducing a desired gene into a rat embryonic stem cell,

(Y) a step for preparing a chimeric rat embryo for transplantation by (i) injecting the rat embryonic stem cell into which the gene was introduced into a blastocoele of a rat blastocyst or a morula stage- or 16-cell stage-rat embryo or (ii) co-culturing the rat embryonic stem cell into which the gene was introduced with two 8-cell stage-rat embryos without zona pellucidas,

(Z) a step for transferring the chimeric rat embryo for transplantation into the uterus of a pseudopregnant female rat to produce the chimeric rat, wherein the rat embryonic stem cell has the following properties (a)-(k);

(a) expressing Oct3/4 gene and Nanog gene,

(b) positive for alkaline phosphatase activity,

(c) having an embryoid body forming ability,

(d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4,

(e) having the same number of chromosomes as does a normal rat cell,

(f) capable of being subcultured and holding the undifferentiated state,

(g) having in vitro pluripotency,

(h) having a potential to differentiate for cells of three embryonic germ lineages,

(i) having teratoma formation ability,

(j) having an ability to produce a chimeric rat,

(k) being germline-competent,

wherein the rat embryonic stem cell is obtained by the method consisting essentially of the following steps (A)-(E) performed using a culture medium with 2% or less serum concentration;

(A) culturing a rat blastocyst on inactivated mouse embryonic fibroblasts in a leukemia inhibitory factor (LIF)-free culture medium to form an inner cell mass in the blastocyst,

(B) dissociating the inner cell mass, wherein the dissociated inner cell mass is in a cell aggregate state,

(C) culturing primary embryonic stem cells resulting from a culture of the dissociated inner cell mass on inactivated mouse embryonic fibroblasts until the primary embryonic stem cells can be passaged,

(D) dissociating the primary embryonic stem cells, which can be passaged, wherein the dissociated primary embryonic stem cells are in a cell aggregate state,

(E) culturing the dissociated primary embryonic stem cells on inactivated mouse embryonic fibroblasts to establish an embryonic stem cell, wherein a rat leukemia inhibitory factor (rLIF)-containing culture medium is used in steps (C)-(E).

3. The method of claim 2, wherein the genetically modified rat is a knockout rat or a knockin rat, and wherein (X) the step for introducing a desired gene into a rat embryonic stem cell comprises introducing a targeting vector into a target gene in the genome of the rat embryonic stem cell by homologous recombination to produce a homologously recombined rat embryonic stem cell.

4. The method of claim 2, wherein the genetically modified rat is a knockdown rat, and wherein (X) the step for introducing a desired gene into a rat embryonic stem cell comprises introducing a vector comprising siRNA or an antisense nucleic acid into the rat embryonic stem cell and expressing the siRNA or antisense nucleic acid to suppress expression of a target gene.